DNA Chain Breaks Research Articles

Sites of gamma radiation-induced DNA strand breaks after alkali treatment.

When DNA is gamma-irradiated in aerated aqueous solution, strand breaks are produced during irradiation or the next few hours. Subsequent piperidine treatment gives rise to further DNA strand ruptures at alkali-labile sites. These different types of DNA chain breaks provoked by gamma-irradiation have been studied with oligonucleotides having defined sequences. The breaks selectively developed inside the DNA chain at alkali-labile sites by piperidine treatment appeared at lower doses preferentially at guanine positions and the order G greater than A greater than T greater than or equal to C was observed. The total contribution of the direct DNA chain ruptures, formed during irradiation and the next few hours, and those obtained by piperidine treatment was studied at doses ranging from 10 to 120 Gy. The chain breaks appeared preferentially at thymine positions and the order T greater than G greater than A greater than or equal to C was shown for the higher doses.

Enhancement of bleomycin-mediated DNA damage by epidermal microsomal enzymes

The role of epidermal microsomal enzymes in catalyzing bleomycin-mediated chain breakage in calf-thymus DNA and in DNA isolated from neonatal rat epidermis was studied. Aerobic incubation of bleomycin with epidermal microsomes, epidermal or calf-thymus DNA and NADPH caused substantial chain breakage of the DNA which was dependent upon concentrations of drug, microsomal protein and NADPH. The reactive oxygen scavenger superoxide dismutase, the metal chelator EDTA, and cytochrome c each inhibited the enzyme-mediated chain breakage reaction. Scavengers of hydrogen peroxide and hydroxyl radicals, including catalase and benzoate and inhibitors of microsomal cytochrome P-450-dependent monooxygenases such as 1-benzylimidazole, metyrapone and α-naphthoflavone, had no inhibitory effects on bleomycin-mediated DNA chain breakage. In contrast, ascorbic acid significantly enhanced DNA damage by bleomycin. These studies indicate that mammalian epidermis possesses membrane-bound enzyme activity capable of enhancing bleomycin-mediated chain breakage of DNA and that oxidation/reduction of adventitious iron and generation of reactive oxygen participate in the reaction. These responses in the epidermis could directly relate to the mechanism of action of intralesional injections of bleomycin which are used quite effectively in the management of recalcitrant human warts. Either epidermal or wart virus DNA or both could be targets for this pharmacologic effect of the drug which is augmented by epidermal microsomal enzymes.

Studies on the interaction of bleomycin A 2 with rat lung microsomes. III. Effect of exogenous iron on bleomycin-mediated DNA chain breakage

The interaction of bleomycin A 2 with rat lung microsomes results in bleomycin-mediated DNA chain breakage due to the mixed-function oxidase catalyzed activation of bleomycin. This study demonstrates that the addition of exogenous Fe 3+ significantly enhances the bleomycin-mediated cleavage of DNA deoxyribose, that the enhancing effect of Fe 3+ is maximum when a 1:1 ratio of bleomycin to Fe 3+ is achieved and that either NADPH or NADH can serve as pyridine cofactors for this reaction. Since the activation of bleomycin can be facilitated by iron in the Fe 2+ form, these observations support the hypothesis that the mixed-function oxidase system may serve to maintain either adventitious or exogenous iron in the Fe 2+ form. In the absence of DNA, the interaction of bleomycin with rat lung microsomes results in the self-inactivation of bleomycin, a reaction which is also enhanced by the addition of exogenous Fe 3+. Thus, the microsomal mixed-function oxidase system represents an efficient biological system for the ‘activation-inactivation’ of bleomycin.

Effect of Heat Shock on Poly(ADP-Ribose) Synthetase and DNA Repair in Drosophila Cells

Poly(ADP-ribose) synthetase, a chromatin-bound enzyme which attaches polyanionic chains of ADP-ribose to nuclear proteins, was found to be temperature sensitive in intact Drosophila melanogaster cells. The synthetase was completely inactivated by heat-shocking the cells at 37/sup 0/C for 5 min, a condition which had no appreciable effect on the subsequent growth of Drosophila cells at their physiological temperature. The heat-shock effect on synthetase was reversible; enzyme activity began to reappear about 2 hr post heat shock. During the 2-hr interval when poly(ADP-ribose) synthetase was absent, the cells were competent in repair of ..gamma..-ray-induced DNA strand breaks as shown by DNA sedimentation studies on alkaline sucrose gradients. It is thus concluded that poly(ADP-ribose) synthesis is unnecessary for repair of DNA strand breaks introduced by irradiation. The same conclusion was reached from the fact that two inhibitors of poly(ADP-ribose) synthetase 3-aminobenzamide and 5-methylnicotinamide, failed to block repair of ..gamma..-ray-induced DNA chain breaks even though both inhibitors reduced the amount of poly(ADP-ribose) synthesized in cells by 50-75%. Although it was found that the repair of DNA strand breaks is independent of poly(ADP-ribose) synthesis, irradiation does activate the synthetase in control cells, as shown by radioimmunoassay of poly(ADP-ribose) levels.

ALTERATION OF URACIL-DNA GLYCOSYLASE ACTIVITY BY URACIL DIMERS IN DNA

— The effects of UV irradiation on double-stranded PBS-2 phage DNA, which contains uracil in place of thymine, were studied. Irradiation by 254 nm light, or by 313 nm light in the presence of the triplet sensitizer acetophenone, resulted in production of uracil-containing pyrimidine photodimers. DNA chain breaks were also produced under both conditions. Uracil-DNA glycosylase released free uracil, but no uracil-containing dimers from such irradiated DNA. The rate of enzymatic release of uracil from irradiated PBS-2 DNA was decreased as the number of photodimers per DNA molecule was increased.

Mapping the topography of DNA wrapped around gyrase by nucleolytic and chemical probing of complexes of unique DNA sequences

Complexes between DNA gyrase and DNA fragments of unique sequences were used to probe the topography of the DNA with nucleases and dimethyl sulfate. The results indicate that the flanking regions, each 50 bp in size, of a 145–155 bp DNA segment resistant to staphylococcal nuclease contain groups of pancreatic DNAase I-susceptible sites that are spaced 10–11 nucleotides apart. Pairs of adjacent DNAase I-sensitive sites on complementary strands are typically staggered by 2–4 bp. The binding of DNA to gyrase confers no protection against alkylation of the DNA by dimethyl sulfate. These properties of the gyrase-DNA complex are reminiscent of those of the nucleosome, and the common underlying structural feature appears to be the wrapping of the DNA around a protein core. The gyrase-DNA complex differs from the nucleosome, however, in that it must possess features necessary for the catalysis of DNA chain breakage and the modulation of the DNA-enzyme interaction by ATP. We present evidence that the breakage and rejoining of the DNA by gyrase occur within a central region of the staphylococcal nuclease-resistant DNA segment. The relation of this observation to the mechanism of DNA supercoiling by gyrase is discussed. Addition of ATP or its β,γ-imido analog has essentially no effect on the patterns of susceptibilities to DNAase I, implying that the DNA-enzyme contacts mapped by the nuclease are little affected by ATP-induced conformational changes.

The effect of bleomycin on prolyl hydroxylase and DNA chain breakage: structure-activity relationship.

The activity of purified prolyl hydroxylase (proline, 2-oxoglutarate dioxygenase, EC 1.14.11.2) was enhanced about 3-fold by addition of bleomycin in the assay mixture. Various members of the bleomycin family, their derivatives and degradation products were investigated for activities against prolyl hydroxylase together with their activities of DNA chain breakage to determine relationships between the structure of bleomycin and its various actions. All the bleomycins with various terminal amine parts and desamide bleomycin stimulated the enzymatic activity but did not exhibit an effect on DNA chain breakage. The stimulatory activity of bleomycin was not decreased by hydrolysis with 0.3 N H2SO4 at 80 degrees C for 6 hours, conditions which liberates the sugar moiety, but was eliminated by hydrolysis with 6 N HCl at 105 degrees C for 24 hours. In contrast both treatments decreased the DNA chain breakage activity of bleomycin. Optical spectral studies revealed that all the bleomycins and their hydrolysates which stimulated the prolyl hydroxylase activity made complexes with ferrous ion, one of the cofactors of this enyzme.

Experimental results with the combination of bleomycin plus mitomycin C.

This investigation has established the following: 1. Bleomycin, in combination with mitomycin C or other quinone-containing anticancer agents, stimulated the damage to KB cells in culture. 2. In AH66 tumor-bearing rats, the simultaneous treatments of bleomycin plus mitomycin C extend the lifespan. 3. The bleomycin-induced DNA chain breakage was enhanced by the NADPH-dependent microsomal electron transport system. The enhancement was also observed at the level of isolated nuclei and cells. Vitamin K2 and mitomycin C increased breakage at the cellular level by bleomycin and NADPH. 4. Bleomycin-Cu2+ had tendency to increase the lipid peroxidation reaction by the microsomes. However, the reaction was effectively inhibited by antioxidants. 5. Bleomycin induced aldehyde formation from DNA breakage. The formation was effectively inhibited by scavenging reactions with hydralazine hydrochloride or isoniazid. The possibility of suppressing the side effect of bleomycin was discussed in relation to TBA reactive compounds.

On the dynamics of DNA in aqueous solution. Pulse radiolysis studies using the light scattering detection method

The kinetics of DNA chain breakage in solution induced by 2 µs pulses of 15 MeV electrons were investigated by light scattering. On irradiating native calf thymus DNA at room temperature the decrease of light scattering intensity (LSI) - due to double strand ruptures - shows a fast decay with a half lifeτ 1/2 of about 30 ms as well as a slow decay withτ 1/2 of about 10 s. With increasing temperature (20–40° C) both the total degree of degradationα and the fraction of the fast decay increase due to the facilitated melting of segments between two single strand breaks on alternate strands forming a double strand break. Above 40° C a third mode of LSI decay withτ 1/2 of 5–10 s arises, indicating detachment of relatively long segments. The total relative decrease of LSI after irradiation A, which can be taken as a measure of the degree of degradationα, follows the square of the absorbed dose in the case of native DNA, whereas on irradiating denatured DNA A rises linearly with dose. The decay of LSI due to the degradation of denatured DNA is much faster than that of native DNA withτ 1/2 down to 150 µs, depending on the absorbed dose. The half lives are interpreted in terms of the separation of fragments by diffusion and of the melting of double strand segments between two single strand breaks.

Role of complex formation in the photosensitized degradation of DNA induced by N'-formylkynurenine.

Abstract— N'‐Formylkynurenine derivatives efficiently bind to DNA or polynucleotides. Homopolynucleotides and DNA display marked differences in the binding process. Association constants are derived which indicate that the oxidized indole ring is more strongly bound to DNA than the unoxidized one. Irradiation of such complexes with wavelengths greater than 320 nm induces pyrimidine dimer formation as well as DNA chain breaks. Complex formation is shown to play an important role in these photosensitized reactions.The photodynamic action of N'‐formylkynurenine on DNA constituents is negligible at neutral pH but guanine and xanthine derivatives are sensitizable at higher pH. Thymine dimer splitting can occur in aggregated frozen aqueous solutions of N'‐formylkynurenine and thymine dimer but this photosensitized splitting is negligible in liquid solutions at room temperature.

Formula omitted] release of thymine from DNA by neocarzinostatin

In vitro degradation of DNA to acid soluble products was induced by the combined action of neocarzinostatin and sulfhydryl agent as 2-mercaptoethanol, dithiothreitol, or reduced glutathione, but not other reducting agent as ascorbic acid or NaBH 4. From the analysis by Sephadex G-10 gel filtration, acid soluble products were found to be thymine and oligonucleotide, but not thymidylic acid and thymidine. Release of adenine or guanine from DNA was not detected. From these results, it is suggested that DNA chain breakage by the combined action of neocarzinostatin and 2-mercaptoethanol may be due to an indirect phosphodiester bond breakage with release of thymine.

Increased DNA chain breakage by combined action of bleomycin and superoxide radical

In vitro DNA chain breakage by bleomycin was enhanced by the addition of xanthine oxidase system. The effect of the xanthine oxidase system disappeared completely when superoxide dismutase was added, but not when catalase was added. From these results it is concluded that superoxide radical is one of the chemical mediators responsible for the enhancement of the DNA chain breakage action of bleomycin.

Dependence upon growth medium and the polA, polC, recA, recB, recC, and exrA genes of separate branches of the uvr Gene-dependent excision-repair process in Escherichia coli K12 cells.

The repair of single-strand breaks which arise in DNA during the uvr gene-dependent excision-repair process was examined in certain radiation-sensitive strains of Escherichia coli K12. The results suggest that the excision-repair process may be divided into two branches, one which is dependent on the presence of growth medium and also the rec+ exr+ genotype, and a second which can occur in buffer (growth medium independent) and is largely dependent on DNA polymerase I. DNA polymerase III appears to be involved in the growth-medium-dependent branch of excision repair, and also in the residual growth-medium-independent repair which occurs in polA1 cells. Chloramphenicol, dinitrophenol, and impurities present in some brands of agar all appear to inhibit the growth-medium-dependent branch of excision-repair. The similarities of the two branches of excision-repair to two known pathways for the repair of X-ray-induced DNA chain breaks are discussed.

DNA content, synthesis and integrity in dividing and non-dividing cells of rec − strains of Escherichia coli K12

Using a method to separate dividing from non-dividing cells of Rec − strains of Escherichia coli K12 (Capaldo & Barbour, 1973), we found: 1. (1) Non-dividing cells of recA −, recB − recC − and recA − recB − recC − strains are defective in DNA synthesis. 2. (2) recA − non-dividing cells contain little or no DNA; recB − recC − and recA − recB − recC − non-dividing cells contain normal amounts of DNA. 3. (3) DNA synthesized in cultures of the recB − recC − and recA − recB − recC − strains is made in the dividing cells and then segregated irreversibly, as a function of subsequent cell division, into the non-dividing cells. 4. (4) Old DNA in recA − recB − recC − cells contains an increased number of single-strand breaks compared to newly synthesized DNA of the same strain or to both old and new DNA from rec + cells; no additional breaks in old DNA of the recB − recC − strain have been detected. We propose that recA +, recB +, recC + gene products are involved in repair of DNA chain breaks that arise during cell growth. According to this view, mutation in the rec genes results in defective repair. This may lead to failure to replicate DNA, loss of viability, degradation of the DNA in the case of the recA − strain, and accumulation of DNA chain breaks in the recA − recB − recC − strain.

Enzymic in vitro repair and chemical nature of DNA chain breaks induced by incorporated phosphorus-32P decay.

In vitro repair of single strand breaks in T4 and phage DNA caused by 32p decay was studied. Zone centrifugation procedure showed that polynucleotide kinase, ligase enzyme system failed to repair 32P-damages. It was found that damaged DNA contained gaps and could be repaired by DNA-polymerase I, polynucleotide ligase treatment.

Inactivation of biologically-active DNA by radiation-induced phenylalanine radicals.

SummaryTo investigate the possibility of the involvement of organic radicals, e.g. those derived from amino acids, in the inactivation of DNA in living cells by ionizing radiation, we studied, as a model system, dilute solutions of the biologically-active DNA of the bacteriophage ΦX174 in the presence of relatively high concentrations of phenylalanine. The solutions were saturated with N2, O2 or N2O and irradiated with 60Co γ-rays. Under these conditions a negligible fraction of the primary water radicals reacts with DNA. Nevertheless inactivation of DNA occurs, due to reactions of amino-acid radicals. These reactions probably do not produce DNA chain breaks, but another type of nucleotide damage, which has not yet been identified. In anoxic solutions the phenylalanine radicals can be reduced or oxidized by small amounts of cysteamine and paranitroacetophenone, respectively, which prevents their attack on DNA, resulting in an enormous protection. Moreover, it was found that the ratio of breaks and other d...